The present invention relates to a factor capable of efficiently regulating expression of an endoplasmic reticulum chaperone gene, a nucleic acid encoding it or a complementary strand nucleic acid thereof, a method for regulating expression of an endoplasmic reticulum chaperone gene, and a method for expressing a foreign gene.
Mammalian cells, like other eukaryotic cells and prokaryotic cells, have developed a number of homeostatic mechanisms to cope with to various physiological and environmental conditions that threaten their survival. Among them, the tightly regulated synthesis of heat shock proteins (HSPs) is a well-known mechanism universally found in all organisms. In addition, the regulated synthesis of glucose-regulated proteins (GRPs), based on a mechanism differing from that of HSP described above, is specifically found in eukaryotic endoplasmic reticulum [Lee, A. S., Curr. Opin. Cell Biol. 4, 267-273 (1992); Morimoto, R. I. et al., The Biology of HEAT SHOCK PROTEINS and MOLECULAR CHAPERONES, Cold Spring Harbor Laboratory (1994)].
In mammals, eight kinds of GRPs, namely, GRP78/Bip, GRP94/ERp99, ORP150/GRP170, ERp72, GRP58/ERp60/ERp61, calreticulin, protein disulfide isomerase (PDI) and FKBP13, have been identified. These GRPs are a series of molecular chaperones or folding enzymes characteristic of the endoplasmic reticulum, each of which expression is induced by the accumulation of proteins that have failed to be folded or have undergone incorrect folding (hereinafter referred to as unfolded proteins) in the endoplasmic reticulum (endoplasmic reticulum stress) [Kozutsumi, Y. et al., Nature 332, 462-464 (1988); Lee, A. S., Trends Biochem. Sci. 12, 20-23 (1987)], and play a very important role in the folding of nascent secretory proteins and membrane proteins in the endoplasmic reticulum. Therefore, these GRPs are hereinafter generically referred to as xe2x80x9cendoplasmic reticulum chaperonesxe2x80x9d.
Expression of endoplasmic reticulum chaperones is also induced by a reagent, such as tunicamycin, which inhibits N-glycosylation of proteins, calcium ionophore A23187, which depletes calcium storage, or thapsigargin, which inhibits calcium-ATPase. These reagents are generally assumed to cause malfunctioning of the endoplasmic reticulum and elicit endoplasmic reticulum stress.
Induction of the above endoplasmic reticulum chaperone by endoplasmic reticulum stress is primarily regulated at the transcription level. Since the endoplasmic reticulum chaperone is not induced by heat shock stress and the promoter sequence of the endoplasmic reticulum chaperone gene contains no heat shock elements, the induction of endoplasmic reticulum chaperone is suggested based on a regulatory mechanism differing from that for the HSP induction. There has yet remain unknown, however, whether an endoplasmic reticulum stress-response is controlled by a common mechanism or by a variety of mechanisms corresponding to individual endoplasmic reticulum chaperones.
The rat GRP78 gene has already been analyzed to some extent, and it is shown that the upstream CORE region and the C1 region comprising the CCAAT sequence are important for transcriptional regulation [Resendez, E. et al., Mol. Cell. Biol. 8, 4579-4584 (1988); Wooden, S. K. et al., Mol. Cell. Biol. 11, 5612-5623 (1991); Li, W. W. et al., Mol. Cell. Biol. 14, 5533-5546 (1994)] (see FIG. 1); however, its transcriptionally regulatory sequence yet remains undetermined. In the case of yeasts, the transcriptionally regulatory sequence (UPRE sequence; CAGNGTG) of the GRP78 gene of the budding yeast is clarified [Mori, K. et al., Genes Cells 1, 803-817 (1996)]. Although a sequence similar to the UPRE sequence is present upstream of the human GRP78 gene, no activity for directing induced transcription by endoplasmic reticulum stress has been detected in any DNA having a sequence similar to that of the UPRE. As mentioned above, the transcriptionally regulatory region involved in an endoplasmic reticulum stress-response of mammals, especially humans, still remains undetermined.
In cancer cells, endoplasmic reticulum chaperones are expressed at high levels. For example, there has been reported that good correlation exists between intracellular GRP78 level and a tumor size [Cai, J. W. et al., J. Cell. Physiol. 154, 229-237 (1993)], and that when GRP78 expression is suppressed by the antisense method, sensitivity to cytotoxic T-cell (CTL) and tumor necrosis factor (TNF) increases [Sugawara, S. et al., Cancer Res. 53, 6001-6005 (1993)] and take is poor in the mouse, and even if taking, it soon results in regression [Jamora, C. et al., Proc. Natl. Acad. Sci. USA 93, 7690-7694 (1996)].
Also, it has been shown that ORP150 is strongly induced in macrophages which infiltrate into arteriosclerotic lesions, and that macrophages treated with an antisense oligonucleotide to suppress ORP150 expression show decreased viability when exposed to hypoxic conditions, especially in the presence of a denatured LDL (low-density lipoprotein) [Tsukamoto, Y. et al., J. Clin. Invest. 98, 1930-1941 (1996)]. Since the macrophages in arteriosclerotic lesions release cytokines, such as tumor necrosis factor, interleukin-1 (IL-1), interleukin-6 (IL-6), fibroblast growth factor (FGF), platelet-derived growth factor (PDGF) and transforming growth factor (TGF-xcex2), the macrophages are positioned at the center of intercellular response in arteriosclerotic lesions and assumed to play a major role in the progression of arteriosclerosis.
Cystic fibrosis is a hereditary disease caused by a mutation of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, the most prevalent of such a mutation being the deletion of phenylalanine at residue 508 (xcex1508F) [Welsh, M. J. and Smith, A. E., Cell 73, 1251-1254 (1993)]. CFTRxcex94508F undergoes abnormal sugar chain addition and is degraded without being transported from the endoplasmic reticulum to the Golgi. However, at low temperatures, such xcex94508F mutants leak from endoplasmic reticulum, are localized in the cell membrane, thereby exhibiting activity [Denning, G. M. et al., Nature 358, 761-764 (1992)]. Appropriately modifying the stringency of quality control for nascent membrane proteins in the endoplasmic reticulum would enable the localization and functioning of CTFRxcex94508F in the cell membrane.
Furthermore, there have been shown that the mRNA of GRP78, as well as of HSP70, is induced in rat cerebral ischemia [Wang, S. et al., Neurochem. Int. 23, 575-582 (1993); Higashi, T. et al., Brain Res. 650, 239-248 (1994)], that the mRNA of GRP78 and GRP94 is induced in the hippocampal dentate gyrus when convulsive seizures are induced with kainic acid [Lowenstein, D. H. et al., Mol. Brain Res. 22, 299-308 (1994); Little, E. et al., Neuroscience 75, 209-219 (1996)], and that ORP150 is induced in the ischemic mouse brain [Kuwabara, K. et al., J. Biol. Chem. 271, 5025-5032 (1996)]. Therefore, the endoplasmic reticulum chaperones are assumed to protectively act on damaged neurocytes by cerebral ischemia or the like.
In addition, in wounded tissues and ulcerative tissues, endoplasmic reticulum chaperones, like HSP, are expected to play an important role in repairing the damaged tissues.
On the other hand, when a foreign gene is introduced into a desired cell, to produce a useful protein, the expression of the introduced gene must be controlled, if the desired gene product exhibits cytotoxicity or affects cell function. In addition, when a foreign, useful protein is expressed in a host using a recombinant DNA, the desired protein in many cases fails to retain the correct conformation so that the protein cannot be expressed at high levels. It is suggested that the amount of the endoplasmic reticulum chaperones and folding enzymes in usual hosts may be insufficient to ensure ample protein expression and formation of correct conformation.
Therefore, there is a demand for a technique for enabling efficient control of the expression of the endoplasmic reticulum chaperones.
An object of the present invention is to provide a method for regulating expression of endoplasmic reticulum chaperone genes, which is capable of increasing or decreasing expression of the above gene; a method for expressing a foreign protein; an endoplasmic reticulum stress transcription factor capable of regulating expression of the above gene; and a nucleic acid encoding it, or a complementary nucleic acid thereof.
The above object and other objects of the present invention will be apparent from the following description.
Concretely, the present invention is concerned with the following:
[1] an endoplasmic reticulum stress transcription factor capable of regulating transcription-inducing activity, wherein the transcription-inducing activity is exhibited by an element having the nucleotide sequence as shown in SEQ ID NO: 1 or an element having a nucleotide sequence having substitution of 1 to 3 bases with other kind of bases in the nucleotide sequence as shown in SEQ ID NO: 1;
[2] a method for controlling expression of an endoplasmic reticulum chaperone, comprising expressing the endoplasmic reticulum stress transcription factor as defined above in item [1];
[3] a method for expressing a foreign protein, comprising positively regulating expression of an endoplasmic reticulum chaperone gene by the method as defined in item [2];
[4] a nucleic acid encoding an activated form of ATF6, or a complementary strand thereto;
[5] a nucleic acid encoding an activated form of CREB-RP, or a complementary strand thereto;
[6] a nucleic acid encoding a suppressive form of ATF6, or a complementary strand thereto; and
[7] a nucleic acid encoding a suppressive form of CREB-RP, or a complementary strand thereto.